Project Summary The neuronal & endothelial nitric oxide (NO) synthase (nNOS & eNOS) enzymes make NO in response to calmodulin (CaM) binding, and function broadly in human health and disease. Posttranslational regulation through phosphorylation further regulates NOS in vivo in response to stimuli. Hallmarks of these large flavo- hemoproteins include multi-domain architecture with flexible linkers, allowing for dynamic, regulated interdomain electron transfer (IET). NO synthesis requires a large conformational change, in which the FMN domain shuttles between NOS's electron-accepting ?input state? and electron-donating ?output state? to deliver electrons across the domains. These large-scale motions are shaped by conformational energy landscape, i.e., the dependence of free energy on protein conformation. Moreover, local conformational adjustment likely continues in the docked state. Despite extensive research efforts, the dynamics underlying these conformational changes required for IET across the NOS domains remain unclear. A roadblock to answering this central question is the lack of a unified theoretical/computational approach to interpret the experimental results quantitatively. Solving this vexing research problem calls for a convergence of mesoscopic computational analysis and hands-on experiments that are sensitive to NOS protein dynamics in solution. Combining these latest experimental methods in a multipronged effort is innovative, as it dramatically expands the overall scope of the experimental measurements and provides a better basis for the computations. This approach will allow us to interpret the diverse experimental results and apply them to the calculated NOS conformational behavior paradigm in a consistent manner. Our integrated program draws on the unique combined expertise of the collaborative team. Importantly, we have made the crucial first step of implementing our experimental and computational approaches synergistically. To determine the energy landscape and the resulting NOS conformational properties, we will first calculate the conformational statistics and dynamics and use it in synergy with the suitable experiments to study long-range tethered domain motions in various NOS proteins. Furthermore, we will investigate local conformational adjustments in the docked state. We will then apply our integrated approach to study remodeling of the conformational landscape by functionally important phosphorylation. Taken together, these results will provide a comprehensive quantitative understanding of protein dynamics as a central part of NOS mechanisms. The proposed research is significant as it will answer long-standing fundamental questions about the NOS isoforms by defining the conformational aspects (statistics, dynamics, and energy landscape) that govern the obligatory electron transfer steps in NOS. This work will positively impact our understanding of other biomolecules as defining structure-dynamics-function relationship lies at the heart of current biochemical research.